The overarching goal of this proposal is to develop novel fluorescence contrast agents that can enhance tumors intraoperatively to guide surgical tumor removal. Nanoparticles used in this proposal will be derived from modifications to hyaluronic acid (HLA). These nanoparticles have the ability entrap near infrared (NIR) fluorescent dyes. Upon entrapment, these NIR fluorescent dyes are quenched. In addition, HLA is a ligand for the cell surface receptor CD44 and is degraded by hyaluronidases. Ideally, specific degradation of HLA-based nanoparticles by hyaluronidases will release free dye that is again fluorescent. Both hyaluronidases and CD44 are overexpressed in a variety of tumors, including invasive ductal carcinoma, which is investigated in this proposal. The HLA-based nanoparticles would comprise a new class of nanoparticle biosensors that specifically identify diseased cells. A key innovative step is that these NIR biosensors can be readily detected by new image-guided surgical instrumentation to provide real-time image-guidance on tumor status. Indeed, an image-guided surgery system has been designed that excites NIR fluorophores with a directed excitation beam. NIR emission is detected spectrally and by CCD detectors in an overhead multicamera detector. The combination of NIR biosensors and intraoperative imaging addresses a key public health concern, the inability to exact complete surgical removal during the first surgery, which can result in a high rate of recurrent tumors. Recurrent tumors, can decrease patient prognosis, change therapeutic regimens, and have severe psychological consequences in addition to the overall costs of additional surgery to health care system. The integration of highly innovative technology to improve patient welfare for those undergoing tumor removal is addressed in four aims: (1) optimize the loading of NIR dye and colloidal properties of NIR dyes through modifying HLA molecular weight, introducing crosslinking chemistry, and coating nanoparticles with poly(ethylene glycol), (2) determine the mechanism for fluorescence activation and CD44 targeting, (3) perform in vivo tumor contrast studies, including image-guided surgery efficacy, and (4) an overall safety evaluation of the agents that includes detailed biodistribution studies, inflammatory and immune system activation in vitro, and blood pharmacokinetics. All in vivo studies will be performed in rodents; mice bearing human breast tumor xenografts, healthy mice, and rats. It's hypothesized that image-guided tumor removal will decrease recurrent disease, while nanoparticle entrapped dye will specifically enhance tumors and their margins resulting in more sensitive cancer detection compared to controls.